Preferentially substituted calcium channel blockers

ABSTRACT

Compounds of the formula  
                 
 
     and their salts,  
     wherein Cy represents cyclohexyl;  
     Y is CH═CHΦ, CHΦ 2 , Φ or Cy,  
     X is trivalent straight-chain alkylene (3-10C) or trivalent straight-chain 1-alkenylene (3-10C) optionally substituted by oxo at the C adjacent N when n is 0 and Y is Φ 2 CH; and is otherwise trivalent straight-chain alkylene (5-10C) or trivalent straight-chain 1-alkenylene (5-10C) optionally substituted by oxo at the C adjacent N;  
     Z is N, NCO, CHNCOR 1  or CHNR 1 , wherein R 1  is alkyl (1-6C); and  
     n is 0-5;  
     wherein each Φ and Cy independently may optionally be substituted by alkyl (1-6C) or by halo, CF 3 , OCF 3 , NO 2 , NR 2 , OR, SR, COR, COOR, CONR 2 , NROCR or OOCR where R is H or alkyl (1-4C), or two substituents may form a 5-7 membered ring  
     with the proviso that the compounds of formula (1) contain at least one aromatic moiety, are useful as calcium channel blockers.

[0001] This application is a continuation-in-part of U.S. Ser. No.09/401,699, filed 23 Sep. 1999 which is a continuation-in-part of U.S.Ser. No. 09/107,037 filed 30 Jun. 1998 and now allowed. The contents ofboth applications are incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention relates to compounds useful in treating conditionsassociated with calcium channel function. More specifically, theinvention concerns compounds containing substituted or unsubstitutedderivatives of 6-membered heterocyclic moieties that are useful intreatment of conditions such as stroke and pain.

BACKGROUND ART

[0003] Native calcium channels have been classified by theirelectrophysiological and pharmacological properties as T, L, N, P and Qtypes (for reviews see McCleskey, E. W. et al. Curr Topics Membr (1991)39:295-326, and Dunlap, K. et al. Trends Neurosci (1995) 18:89-98).T-type (or low voltage-activated) channels describe a broad class ofmolecules that transiently activate at negative potentials and arehighly sensitive to changes in resting potential. The L, N, P and Q-typechannels activate at more positive potentials (high voltage activated)and display diverse kinetics and voltage-dependent properties. There issome overlap in biophysical properties of the high voltage-activatedchannels, consequently pharmacological profiles are useful to furtherdistinguish them. L-type channels are sensitive to dihydropyridineagonists and antagonists, N-type channels are blocked by the Conusgeographus peptide toxin, ω-conotoxin GVIA, and P-type channels areblocked by the peptide ω-agatoxin IVA from the venom of the funnel webspider, Agelenopsis aperta. A fourth type of high voltage-activatedcalcium channel (Q-type) has been described, although whether the Q- andP-type channels are distinct molecular entities is controversial(Sather, W. A. et al. Neuron (1995) 11:291-303; Stea, A. et al. ProcNatl Acad Sci USA (1994) 91:10576-10580; Bourinet, E. et al. NatureNeuroscience (1999) 2:407-415). Several types of calcium conductances donot fall neatly into any of the above categories and there isvariability of properties even within a category suggesting thatadditional calcium channels subtypes remain to be classified.

[0004] Biochemical analyses show that neuronal high voltage activatedcalcium channels are heterooligomeric complexes consisting of threedistinct subunits (α₁, α₂δ and β) (reviewed by De Waard, M. et al. IonChannels (1997) vol. 4, Narahashi, T. ed. Plenum Press, NY). The α₁subunit is the major pore-forming subunit and contains the voltagesensor and binding sites for calcium channel antagonists. The mainlyextracellular α₂ is disulfide-linked to the transmembrane δ subunit andboth are derived from the same gene and are proteolytically cleaved invivo. The β subunit is a nonglycosylated, hydrophilic protein with ahigh affinity of binding to a cytoplasmic region of the α₁ subunit. Afourth subunit, γ, is unique to L-type calcium channels expressed inskeletal muscle T-tubules. The isolation and characterization ofγ-subunit-encoding cDNAs is described in U.S. Pat. No. 5,386,025 whichis incorporated herein by reference.

[0005] Recently, each of these α₁ subtypes has been cloned andexpressed, thus permitting more extensive pharmacological studies. Thesechannels have been designated α_(1A)-α_(1I) and α_(1S) and correlatedwith the subtypes set forth above. α_(1A) channels are of the P/Q type;α_(1B) represents N; α_(1C), α′_(1D), α_(1F) and α_(1S) represent L;α_(1E) represents a novel type of calcium conductance, and α_(1G)-α_(1I)represent members of the T-type family, reviewed in Stea, A. et al. inHandbook of Receptors and Channels (1994), North, R. A. ed. CRC Press;Perez-Reyes, et al. Nature (1998) 391:896-900; Cribbs, L. L. et al.Circulation Research (1998) 83:103-109; Lee, J. H. et al. Journal ofNeuroscience (1999) 19:1912-1921.

[0006] Further details concerning the function of N-type channels, whichare mainly localized to neurons, have been disclosed, for example, inU.S. Pat. No. 5,623,051, the disclosure of which is incorporated hereinby reference. As described, N-type channels possess a site for bindingsyntaxin, a protein anchored in the presynaptic membrane. Blocking thisinteraction also blocks the presynaptic response to calcium influx.Thus, compounds that block the interaction between syntaxin and thisbinding site would be useful in neural protection and analgesia. Suchcompounds have the added advantage of enhanced specificity forpresynaptic calcium channel effects.

[0007] U.S. Pat. No. 5,646,149 describes calcium channel antagonists ofthe formula A-Y-B wherein B contains a piperazine or piperidine ringdirectly linked to Y. An essential component of these molecules isrepresented by A, which must be an antioxidant; the piperazine orpiperidine itself is said to be important. The exemplified compoundscontain a benzhydril substituent, based on known calcium channelblockers (see below). U.S. Pat. No. 5,703,071 discloses compounds saidto be useful in treating ischemic diseases. A mandatory portion of themolecule is a tropolone residue; among the substituents permitted arepiperazine derivatives, including their benzhydril derivatives. U.S.Pat. No. 5,428,038 discloses compounds which are said to exert a neuralprotective and antiallergic effect. These compounds are coumarinderivatives which may include derivatives of piperazine and othersix-membered heterocycles. A permitted substituent on the heterocycle isdiphenylhydroxymethyl. Thus, approaches in the art for variousindications which may involve calcium channel blocking activity haveemployed compounds which incidentally contain piperidine or piperazinemoieties substituted with benzhydril but mandate additional substituentsto maintain functionality.

[0008] Certain compounds containing both benzhydril moieties andpiperidine or piperazine are known to be calcium channel antagonists andneuroleptic drugs. For example, Gould, R. J. et al. Proc Natl Acad SciUSA (1983) 80:5122-5125 describes antischizophrenic neuroleptic drugssuch as lidoflazine, fluspirilene, pimozide, clopimozide, andpenfluridol. It has also been shown that fluspirilene binds to sites onL-type calcium channels (King, V. K. et al. J Biol Chem (1989)264:5633-5641) as well as blocking N-type calcium current (Grantham, C.J. et al. Brit J Pharmacol (1944) 111:483-488). In addition, Lomerizine,as developed by Kanebo KK, is a known calcium channel blocker;Lomerizine is, however, not specific for N-type channels. A review ofpublications concerning Lomerizine is found in Dooley, D., CurrentOpinion in CPNS Investigational Drugs (1999) 1:116-125.

[0009] The present invention is based on the recognition that thecombination of a six-membered heterocyclic ring containing at least onenitrogen said nitrogen coupled through a linker to a benzhydril moietyresults in effective calcium channel blocking activity. In some casesenhanced specificity for N-type channels, or decreased specificity forL-type channels is shown. The compounds are useful for treating strokeand pain and other calcium channel-associated disorders, as furtherdescribed below. By focusing on these moieties, compounds useful intreating indications associated with calcium channel activity areprepared.

DISCLOSURE OF THE INVENTION

[0010] The invention relates to compounds useful in treating conditionssuch as stroke, head trauma, migraine, chronic, neuropathic and acutepain, epilepsy, hypertension, cardiac arrhythmias, and other indicationsassociated with calcium metabolism, including synaptic calciumchannel-mediated functions. The compounds of the invention arebenzhydril or partly saturated benzhydril derivatives of piperidine orpiperazine with substituents which enhance the calcium channel blockingactivity of the compounds. Thus, in one aspect, the invention isdirected to therapeutic methods that employ compounds of the formula

[0011] wherein Cy represents cyclohexyl;

[0012] Y is CH═CHΦ, CHΦ₂, Φ or Cy,

[0013] X is trivalent straight-chain alkylene (3-10C) or trivalentstraight-chain 1-alkenylene (3-10C) optionally substituted by oxo at theC adjacent N when n is 0 and Y is Φ₂CH; and is otherwise trivalentstraight-chain alkylene (5-10C) or trivalent straight-chain 1-alkenylene(5-10C) optionally substituted by oxo at the C adjacent N;

[0014] Z is N, NCO, CHNCOR¹ or CHNR¹, wherein R¹ is alkyl (1-6C); and

[0015] n is 0-5;

[0016] wherein each Φ and Cy independently may optionally be substitutedby alkyl (1-6C) or by halo, CF₃, OCF₃, NO₂, NR₂, OR, SR, COR, COOR,CONR₂, NROCR or OOCR where R is H or alkyl (1-4C), or two substituentsmay form a 5-7 membered ring

[0017] with the proviso that the compounds of formula (1) contain atleast one aromatic moiety.

[0018] The invention is directed to methods to antagonize calciumchannel activity using the compounds of formula (1) and thus to treatassociated conditions. It will be noted that the conditions may beassociated with abnormal calcium channel activity, or the subject mayhave normal calcium channel function which nevertheless results in anundesirable physical or metabolic state. In another aspect, theinvention is directed to pharmaceutical compositions containing thesecompounds.

[0019] The invention is also directed to combinatorial librariescontaining the compounds of formula (1) and to methods to screen theselibraries for members containing particularly potent calcium channelblocking activity or for members that antagonize one type of suchchannels specifically.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows a comparison of certain preferred embodiments of thecompounds of the invention to the known compound Lomerizine.

[0021]FIG. 2 is a graphic representation of the specificity of thecompounds shown in FIG. 1 with respect to N-type, L-type and P/Q-typechannels.

[0022]FIG. 3 is a graphic representation of the data shown in FIG. 2based on IC₅₀ values calculated from the data shown in FIG. 2.

MODES OF CARRYING OUT THE INVENTION

[0023] The compounds of formula (1), useful in the methods of theinvention, exert their desirable effects through their ability toantagonize the activity of calcium channels. This makes them useful fortreatment of certain conditions. Among such conditions are stroke,epilepsy, head trauma, migraine and chronic, neuropathic and acute pain.Calcium flux is also implicated in other neurological disorders such asschizophrenia, anxiety, depression, other psychoses, and certaindegenerative disorders. Other treatable conditions includecardiovascular conditions such as hypertension and cardiac arrhythmias.

[0024] While the compounds of formula (1) generally have this activity,the availability of a multiplicity of calcium channel blockers permits anuanced selection of compounds for particular disorders. Thus, theavailability of this class of compounds provides not only a genus ofgeneral utility in indications that are affected by excessive calciumchannel activity, but also provides a large number of compounds whichcan be mined and manipulated for specific interaction with particularforms of calcium channels. The availability of recombinantly producedcalcium channels of the α_(1A)-α_(1I) and α_(1S) types set forth above,facilitates this selection process. Dubel, S. J. et al. Proc Natl AcadSci USA (1992) 89:5058-5062; Fujita, Y. et al. Neuron (1993) 10:585-598;Mikami, A. et al. Nature (1989) 340:230-233; Mori, Y. et al. Nature(1991) 350:398-402; Snutch, T. P. et al. Neuron (1991) 7:45-57; Soong,T. W. et al. Science (1993) 260:1133-1136; Tomlinson, W. J. et al.Neuropharmacology (1993) 32:1117-1126; Williams, M. E. et al. Neuron(1992) 8:71-84; Williams, M. E. et al. Science (1992) 257:389-395;Perez-Reyes, et al. Nature (1998) 391:896-900; Cribbs, L. L. et al.Circulation Research (1998) 83:103-109; Lee, J. H. et al. Journal ofNeuroscience (1999) 19:1912-1921.

[0025] Thus, while it is known that calcium channel activity is involvedin a multiplicity of disorders, the types of channels associated withparticular conditions is the subject of ongoing data collection. Forexample, the association of N-type channels in conditions associatedwith neural transmission would indicate that compounds of the inventionwhich target N-type receptors are most useful in these conditions. Manyof the members of the genus of compounds of formula (1) exhibit highaffinity for N-type channels; other members of the genus maypreferentially target other channels.

[0026] There are two distinguishable types of calcium channelinhibition. The first, designated “open channel blockage,” isconveniently demonstrated when displayed calcium channels are maintainedat an artificially negative resting potential of about −100 mV (asdistinguished from the typical endogenous resting maintained potentialof about −70 mV). When the displayed channels are abruptly depolarizedunder these conditions, calcium ions are caused to flow through thechannel and exhibit a peak current flow which then decays. Open channelblocking inhibitors diminish the current exhibited at the peak flow andcan also accelerate the rate of current decay.

[0027] This type of inhibition is distinguished from a second type ofblock, referred to herein as “inactivation inhibition.” When maintainedat less negative resting potentials, such as the physiologicallyimportant potential of −70 mV, a certain percentage of the channels mayundergo conformational change, rendering them incapable of beingactivated—i.e., opened—by the abrupt depolarization. Thus, the peakcurrent due to calcium ion flow will be diminished not because the openchannel is blocked, but because some of the channels are unavailable foropening (inactivated). “Inactivation” type inhibitors increase thepercentage of receptors that are in an inactived state.

Synthesis

[0028] The compounds of the invention may be synthesized usingconventional methods. Illustrative of such methods are Schemes 1 and 2:

[0029] Alternatively, a carboxylic acid containing the benzhydril (orΦCyCH or Cy₂CH) moiety can first be synthesized and then reacted withthe piperazine (or piperidine) moiety and subsequently reduced. Tosynthesize the desired acid, an ω-bromo carboxylic acid is refluxedwith, in the case of benzhydril, triphenylphosphine in the presence ofmethyl nitrile and then treated with lithium hexamethyldisilazide in asolvent such as THF. The resulting unsaturated carboxylic acidcontaining the two phenyl substituents is then reduced as shown inScheme 1 with hydrogen on a palladium catalyst and then reacted withderivatized piperazine (or piperidine) to form the amide. The amide canthen be reduced as shown above.

Preferred Embodiments

[0030] The compounds of formula (1) are defined as shown in terms of theembodiments of their various substituents:

[0031] Particularly preferred embodiments of the compounds of formula(1) are those wherein X is coupled to two phenyl groups. Less preferredare instances where X is coupled to one phenyl and one cyclohexyl. Leastpreferred are those instances wherein X is coupled to two cyclohexylgroups.

[0032] As defined above, X may be a trivalent straight-chain alkylene of5-10C optionally substituted with oxo at the position adjacent thepiperidine or piperazine ring nitrogen. Preferably, the alkylene chainis 5-8C, more preferably 5-7C, and even more preferably 5-6C.Substitution with oxo is preferred only when the length of the alkylenechain is 6-10C. In addition, X may be a straight-chain 1-alkenylene(5-10C) wherein the π-bond is in the position distal to the piperidineor pyrimidine ring nitrogen. Under these circumstances, the two cyclicmoieties are accommodated by the alkenylene chain by virtue of thealkenylene chain as a vinyl substituent to each cyclic moiety. Inaddition, when n is 0 and Y is Φ₂CH, the embodiment of X described abovemay also be shorter and may contain 3-10C.

[0033] Preferred embodiments of Z are N, NCO and CHNR¹ where R¹ ispreferably H but may also be alkyl (1-6C), preferably 1-4C, morepreferably 1-2C, and even more preferably methyl (or H).

[0034] Preferred embodiments for n are 0-4, more preferably 1-2.

[0035] Any of the phenyl or cyclohexyl moieties contained in thecompounds of formula (1) may be substituted, as noted above. Preferredsubstituents include halo, especially fluoro, NO₂, alkyl (1-6C),preferably methyl, OR, preferably methoxy, NR₂, preferablydimethylamino, diethylamino, methylamino or ethylamino, acetamido, CF₃,OCF₃ and the like. Two substituted positions may also form a ring.Preferably, where the cyclic moieties coupled to X are both phenyl, thephenyl groups are identically substituted. Where one such moiety isphenyl and the other is cyclohexyl, the presence of a substituent on thephenyl moiety and an unsubstituted cyclohexyl moiety are preferred. Itis believed that halogenation of the compounds of the invention ishelpful in modulating the in vivo half-life, and it may be particularlyadvantageous to include halogen substituents, such as fluorosubstitutions on any phenyl moieties.

[0036] Particularly preferred are compounds MC-34D, JM-G-10, 39-1-B4,and 39-45-3 shown in FIG. 1 and variously substituted forms thereof.

[0037] Thus, also preferred are forms of these enumerated compoundswhich contain different substituents on the phenyl or cyclohexylmoieties from those shown. Thus, also preferred are compounds having thegeneral formula of MC-34D wherein the two phenyl moieties attached to Xcontain fluoro in the para position. Alternative substitutions are asshown below where Φ1 and Φ2 indicate the 2 phenyl groups attached to X(the numbers being arbitrarily chosen as these phenyl groups areequivalent) and Φ3 represents phenyl group contained in Y. In addition,also preferred are embodiments as set forth herein where Z of MC-34D isNCO or wherein X is —CH(CH₂)-₅. Φ1 Φ2 Φ3 2,4-dimethyl 2,4,dimethyl 4-F4-methoxy 3-chloro 4-methyl 2,4,6 trimethyl 2,4,6 trimethyl — aminoamino 4-F 4-F 4-F 4-F 4-F 4-methoxy 4-F 4-F 3,4-OCH₂O—

[0038] Similarly, JM-G-10 with substituents on the phenyl groups andcyclohexyl group may be employed in the methods of the invention,preferred embodiments also include those wherein X is —CH(CH₂)-₅.Suitable substitutions are shown below: Φ1 Φ2 Cy 4-F 4-F — 2,4-dimethoxy4-methyl 3,5-diethyl 3,5-diamino 3;5-diamino 4-F

[0039] Alternatively substituted compounds of formula 39-1-B4 are alsoincluded within the preferred embodiments of the invention. In the tablebelow, Φ1 and Φ2 represent the two equivalent phenyl groups coupled to Xand Φ3 and Φ4 represent the two equivalent phenyl groups included in Y.Forms of 39-1-B4 wherein the carbonyl group in X is reduced to methyleneare also preferred, including those with the substituents shown below.Φ1 Φ2 Φ3 Φ4 4-F 4-F — — 3,4-CH₂CH₂CH₂— 3,4-CH₂CH₂CH₂— 4-F 4-F2,6-dimethoxy 3,5,diamino 3,5,diamino 2,6,dimethoxy 4-COOH 4-COOH 3,5dichloro 3,5-dichloro

[0040] Similarly, various alternative substitution patterns on compound39-45-3 may be employed. Included are those embodiments where a carbonylis present adjacent the piperazine in the substituent X. Also includedare analogs where n=0. Particularly preferred are embodiments where twosubstituents on the phenyl group contained in Y form a ring,particularly a 5-membered ring. Thus, preferred substitution patternsare those set forth below where Φ1 and Φ2 represent the two equivalentphenyl groups attached to X and Φ3 represents the phenyl group containedin Y. Φ1 Φ2 Φ3 4-F 4-F 3,4,5-trimethoxy 4-F 4-F 3,4,-CH2 CH2—CH2— 4-F4-F 3,4,5-tri trifluro-methyl 4-F 4-F 3,4,-OCH2O— 2,4,6 trimethoxy — 4-F3,5-diethoxy 3,5-diethoxy 3,5-diethoxy 4-F 4-F 3,5-detrifluoromethyl 4-F4-F 5-OCF3 4-f 4-F 3,4 dimethyl 4-F 4-F 3-methoxy 4-F 4-F 4-F 4-F 4-F3-methyl 4-F 4-F 2-methoxy 4-F 4-F 4-acetyl

[0041] The pattern of substitution will influence the strength ofcalcium channel blocking ability as well as specificity.

[0042] Where the structure permits, invention compounds may also besupplied as pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts include the acid addition salts which can be formedfrom inorganic acids such as hydrochloric, sulfuric, and phosphoric acidor from organic acids such as acetic, propionic, glutamic, glutaric, aswell as acid ion-exchange resins.

Libraries and Screening

[0043] The compounds of the invention can be synthesized individuallyusing methods known in the art per se, or as members of a combinatoriallibrary.

[0044] Synthesis of combinatorial libraries is now commonplace in theart. Suitable descriptions of such syntheses are found, for example, inWentworth, Jr., P. et al. Current Opinion in Biol (1993) 9:109-115;Salemme, F. R. et al. Structure (1997) 5:319-324. The libraries containcompounds with various substitutents and various degrees ofunsaturation, as well as different chain lengths. The libraries, whichcontain, as few as 10, but typically several hundred members to severalthousand members, may then be screened for compounds which areparticularly effective against a specific subtype of calcium channel,i.e., the N-type channel. In addition, using standard screeningprotocols, the libraries may be screened for compounds which blockadditional channels or receptors such as sodium channels, potassiumchannels and the like.

[0045] Methods of performing these screening functions are well known inthe art. Typically, the receptor to be targeted is expressed at thesurface of a recombinant host cell such as human embryonic kidney cells.The ability of the members of the library to bind the channel to betested is measured, for example, by the ability of the compound in thelibrary to displace a labeled binding ligand such as the ligand normallyassociated with the channel or an antibody to the channel. Moretypically, ability to antagonize the receptor is measured in thepresence of calcium ion and the ability of the compound to interferewith the signal generated is measured using standard techniques.

[0046] In more detail, one method involves the binding of radiolabeledagents that interact with the calcium channel and subsequent analysis ofequilibrium binding measurements including, but not limited to, onrates, off rates, K_(d) values and competitive binding by othermolecules. Another method involves the screening for the effects ofcompounds by electrophysiological assay whereby individual cells areimpaled with a microelectrode and currents through the calcium channelare recorded before and after application of the compound of interest.Another method, high-throughput spectrophotometric assay, utilizesloading of the cell lines with a fluorescent dye sensitive tointracellular calcium concentration and subsequent examination of theeffects of compounds on the ability of depolarization by potassiumchloride or other means to alter intracellular calcium levels.

[0047] As described above, a more definitive assay can be used todistinguish inhibitors of calcium flow which operate as open channelblockers, as opposed to those that operate by promoting inactivation ofthe channel. The methods to distinguish these types of inhibition aremore particularly described in the examples below. In general,open-channel blockers are assessed by measuring the level of peakcurrent when depolarization is imposed on a background resting potentialof about −100 mV in the presence and absence of the candidate compound.Successful open-channel blockers will reduce the peak current observedand may accelerate the decay of this current. Compounds that areinactivated channel blockers are generally determined by their abilityto shift the voltage dependence of inactivation towards more negativepotentials. This is also reflected in their ability to reduce peakcurrents at more depolarized holding potentials (e.g., −70 mV) and athigher frequencies of stimulation, e.g., 0.2 Hz vs. 0.03 Hz.

Utility and Administration

[0048] For use as treatment of human and animal subjects, the compoundsof the invention can be formulated as pharmaceutical or veterinarycompositions. Depending on the subject to be treated, the mode ofadministration, and the type of treatment desired—e.g., prevention,prophylaxis, therapy; the compounds are formulated in ways consonantwith these parameters. A summary of such techniques is found inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCo., Easton, Pa., incorporated herein by reference.

[0049] In general, for use in treatment, the compounds of formula (1)may be used alone, as mixtures of two or more compounds of formula (1)or in combination with other pharmaceuticals. Depending on the mode ofadministration, the compounds will be formulated into suitablecompositions to permit facile delivery.

[0050] Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. The compounds can be administered also inliposomal compositions or as microemulsions.

[0051] For injection, formulations can be prepared in conventional formsas liquid solutions or suspensions or as solid forms suitable forsolution or suspension in liquid prior to injection or as emulsions.Suitable excipients include, for example, water, saline, dextrose,glycerol and the like. Such compositions may also contain amounts ofnontoxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents and the like, such as, for example, sodium acetate,sorbitan monolaurate, and so forth.

[0052] Various sustained release systems for drugs have also beendevised. See, for example, U.S. Pat. No. 5,624,677.

[0053] Systemic administration may also include relatively noninvasivemethods such as the use of suppositories, transdermal patches,transmucosal delivery and intranasal administration. Oral administrationis also suitable for compounds of the invention. Suitable forms includesyrups, capsules, tablets, as in understood in the art.

[0054] For administration to animal or human subjects, the dosage of thecompounds of the invention is typically 0.1-15 mg/kg, preferably 0.1-1mg/kg. However, dosage levels are highly dependent on the nature of thecondition, the condition of the patient, the judgment of thepractitioner, and the frequency and mode of administration.

[0055] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 Assessment of Calcium Channel Blocking Activity

[0056] Antagonist activity was measured using whole cell patchrecordings on human embryonic kidney cells either stably or transientlyexpressing rat α_(1B)+α_(2b)+β_(1b) channels (N-type channels) with 5 mMbarium as a charge carrier.

[0057] For transient expression, host cells, such as human embryonickidney cells, HEK 293 (ATCC# CRL 1573) were grown in standard DMEMmedium supplemented with 2 mM glutamine and 10% fetal bovine serum. HEK293 cells were transfected by a standard calcium-phosphate-DNAcoprecipitation method using the rat α_(1B)+β_(1b)+α₂δ N-type calciumchannel subunits in a vertebrate expression vector (for example, seeCurrent Protocols in Molecular Biology).

[0058] After an incubation period of from 24 to 72 hrs the culturemedium was removed and replaced with external recording solution (seebelow). Whole cell patch clamp experiments were performed using anAxopatch 200B amplifier (Axon Instruments, Burlingame, Calif.) linked toan IBM compatible personal computer equipped with pCLAMP software.Borosilicate glass patch pipettes (Sutter Instrument Co., Novato,Calif.) were polished (Microforge, Narishige, Japan) to a resistance ofabout 4 MΩ when filled with cesium methanesulfonate internal solution(composition in MM: 109 CsCH₃SO₄, 4 MgCl₂, 9 EGTA, 9 HEPES, pH 7.2).Cells were bathed in 5 mM Ba⁺⁺ (in mM: 5 BaCl₂, 1 MgCl₂, 10 HEPES, 40tetraethylammonium chloride, 10 glucose, 87.5 CsCl pH 7.2). Current datashown were elicited by a train of 100 ms test pulses at 0.066 Hz from−100 mV and/or −80 mV to various potentials (min. −20 mV, max. +30 mV).Drugs were perfused directly into the vicinity of the cells using amicroperfusion system.

[0059] Normalized dose-response curves were fit (Sigmaplot 4.0, SPSSInc., Chicago, Ill.) by the Hill equation to determine IC₅₀ values.Steady-state inactivation curves were plotted as the normalized testpulse amplitude following 5 s inactivating prepulses at +10 mVincrements. Inactivation curves were fit (Sigmaplot 4.0) with theBoltzman equation, I_(peak) (normalized)=1/(1+exp((V−V_(h))z/25.6)),where V inactivation potentials, respectively, and z is the slopefactor.

EXAMPLE 2 Synthesis of Illustrative Compounds of Formula (1)

[0060] A. Synthesis of 6,6-Diphenyl Hexanoic Acid.

[0061] 6-Bromohexanoic acid (7.08 g, 36.3 mmole) and triphenylphosphine(10 g, 38.2 mmole) were mixed in dry CH₃CN (40 ml), heated to refluxovernight and allowed to cool to RT. The solution was concentrated underreduced pressure to give a viscous gel. Approximately 75 ml of THF wasadded to the reaction mixture and the walls of the flask were scratchedwith a spatula to start crystallization. The resulting solid wasfiltered under vacuum, washed with THF and dried under reduced pressureand used without further purification.

[0062] This product (1.5 g) was suspended in dry THF (10 ml) and theflask purged with N₂ and cooled to −78° C. To the stirred reaction wasadded lithium hexamethyldisilazide (LiHMDS) (10 ml, 1M in THF). Theyellow solution was stirred at −78° C. for 1 h over which time thereaction darkened slightly. The cooling bath was removed and thereaction allowed to warm to RT. The reaction was kept at RT for 1 hduring which time the solution turned a dark red color and most of thesolids went into solution. Benzophenone (0.54 g in 3 ml THF) was addedto the reaction and allowed to react overnight. The yellow solution wasconcentrated under reduced pressure to give a yellow solid. Theresulting solid was partitioned between ether and 10% HCl. The organiclayer was washed with water (2×) and extracted with 10% NaOH (3×). Thecombined aqueous base fraction was acidified with conc. HCl to a pH of4. The water layer was extracted with ether (3×) and the combinedorganic fractions dried over Na₂SO₄.

[0063] The ether was evaporated to dryness under reduced pressure togive a colorless oil which crystallized on standing to give a waxysolid, 6,6-diphenyl hex-5-enoic acid, which was dissolved in 30 ml MeOHand mixed with 5% Pd—C and placed in a Parr hydrogenator. The reactionvessel was purged with hydrogen and pressurized to 60 PSIG and reactedat RT for 4 h. The reaction mixture was sampled and analyzed by TLC. Ifthe TLC when stained with KMnO₄ showed a positive test for alkenes thereaction mixture was resubjected to the reaction conditions. Thesolution was then filtered through a plug of celite and the methanolfiltrate containing 6,6-diphenyl hexanoic acid was concentrated undervacuum.

[0064] B. Reaction with Substituted Piperazine.

[0065] 6,6-Diphenylhexanoic acid (0.4 mmoles) was mixed with the desiredsubstituted piperazine (0.35 mmoles) in dry THF (7 ml). EDC (0.5 mmoles)and DMAP (cat) were added and the mixture heated to 40° C. with shakingovernight. The reaction was diluted with ethyl acetate and washed withwater (4×) and 10% NaOH (3×) and dried over sodium sulfate andevaporated to dryness. The resulting residue was purified by columnchromatography (silica gel, 1:1 hexane:EtOAc), and the products werecharacterized by HPLC-MS.

[0066] Piperazines used in the foregoing procedure includephenylpiperazine, benzylpiperazine, benzhydrilpiperazine, and piperazinesubstituted at the 1-position with Φ-CH═CH₂—.

[0067] The resulting compounds contain a carbonyl adjacent to the ringnitrogen of piperazine. These compounds are of formula (1) and exhibitcalcium channel blocking activity.

[0068] C. Reduction of CO.

[0069] The compounds prepared in paragraph B were dissolved in dry THF(5 ml) and reacted with LiAlH₄ (1M in THF) and allowed to react for 6 h.The reactions were quenched with EtOAc (15 ml) and extracted with water(5×) 10% NaOH (10×), brine (1×), dried over sodium sulfate andconcentrated under reduced pressure. Most of the products at this stagewere >80% pure. Those <80% were purified for running a short column(silica gel, 1:1 hex:EtOAc).

EXAMPLE 3 Preparation of Compounds of Formula (1) fromBenzhydrilpiperazine Derivatives

[0070] N-(Diphenylmethyl)piperazine (0.5 mmole) was dissolved in dry THF(10 ml). To each reaction flask was added powdered K₂CO₃ and acidchloride of the formula Y—CO—Cl (0.7 mmole). The reaction was stirred atRT for 2 h and quenched with 105 NaOH (10 ml) and extracted with EtOAc(10 ml). The organic layer was washed with 10% NaOH (4×) and dried oversodium sulfate, concentrated, and purified by column chromatography(silica gel, 1:1 hex:EtOAc) to give the desired amide. Acyl halides usedin this procedure included cyclohexyl COCl, ΦCOCl and ΦCH═CHCOCl.

[0071] To reduce the resulting amide, the above product was dissolved indry THF (5 ml) and reacted with LiAlH₄ (1M in THF) and allowed to reactfor 6 h. The reaction was quenched with EtOAc (15 ml) and extracted withwater (5×) 10% NaOH (10×), brine (1×), dried over sodium sulfate andconcentrated under reduced pressure. Most of the products at this stagewere >80% pure. Those <80% were purified for running a short column(silica gel, 1:1 hex:EtOAc).

EXAMPLE 4 Channel Blocking Activities of Various Invention Compounds

[0072] Using the procedure set forth in Example 1, various compounds ofthe invention were tested for their ability to block N-type calciumchannels. The results are shown in the table below where IC₅₀ is givenin μM (micromolar). X coupled to X Z n Y IC₅₀ Φ, Φ CHCH₂ CHNH 1 Φ ±3 Φ,Φ CH(CH₂)₂ CHNH 1 Φ 2 Φ, Φ CHCH₂ CHNH 1 Cy 3-4 Φ, Φ CH(CH₂)₂ CHNH 1 Cy2-3 Φ, Φ CH(CH₂)₄CO CHNH 1 Cy 0.75 Φ, Φ C═CH(CH₂)₂ N 1 Cy 5.2 Φ, CyCH(CH₂)₅ N 1 CH═CHΦ 5.9 Φ, Cy CH(CH₂)₄CO N 1 CH═CHΦ 3.9 Φ, Cy CH(CH₂)₅CON 1 CH═CHΦ 3.2 Φ, Cy CH(CH₂)₅CO N 0 CHΦ₂ 10.2 Φ, Cy CH(CH₂)₅CO N 1CH═CHΦ 12.2 Φ, Cy CH(CH₂)₆ N 1 Cy 7.2 Φ, Cy CHCH₂ N 1 Cy 20.2 Φ, Cy CHCON 1 Φ 14.2 Φ, Cy CHCO N 1 CH═CHΦ 5.9 Φ, Cy CHCH₂ N 1 Φ ±5 Φ, Cy CH(CH₂)₅N 2 Φ 3.1 Φ, Cy CHCO N 1 CH═CHΦ ±5 Φ, Cy CHCH₂ N 1 CH═CHΦ 10.6 Φ, CyCHCH₂ N 1 CH═CHΦ ±5 Φ, Cy CHCO N 1 Φ 20 Φ, Cy CHCO N 1 CHΦ₂ 35 Φ, CyCHCH₂ N 1 CHΦ₂ 20

EXAMPLE 5 Additional Methods

[0073] The methods of Examples 1 and 2 were followed with slightmodifications as will be apparent from the description below.

[0074] A. Transformation of HEK Cells:

[0075] N-type calcium channel blocking activity was assayed in humanembryonic kidney cells, HEK 293, stably transfected with the rat brainN-type calcium channel subunits (α_(1B)+α_(2δ)+β_(1b) cDNA subunits).Alternatively, N-type calcium channels (α_(1B)+α_(2δ)+β_(1b) cDNAsubunits), L-type channels (α_(1C)+α_(2δ)+β_(1b) cDNA subunits) andP/Q-type channels (α_(1A)+α_(2δ)+β_(1b) cDNA subunits) were transientlyexpressed in HEK 293 cells. Briefly, cells were cultured in Dulbecco'smodified eagle medium (DMEM) supplemented with 10% fetal bovine serum,200 U/ml penicillin and 0.2 mg/ml streptomycin at 37° C. with 5% CO₂. At85% confluency cells were split with 0.25% trypsin/1 mM EDTA and platedat 10% confluency on glass coverslips. At 12 hours the medium wasreplaced and the cells transiently transfected using a standard calciumphosphate protocol and the appropriate calcium channel cDNAs. Fresh DMEMwas supplied and the cells transferred to 28° C./5% CO₂. Cells wereincubated for 1 to 2 days to whole cell recording.

[0076] B. Measurement of Inhibition:

[0077] Whole cell patch clamp experiments were performed using anAxopatch 200B amplifier (Axon Instruments, Burlingame, Calif.) linked toa personal computer equipped with pCLAMP software. The external andinternal recording solutions contained, respectively, 5 mM BaCl₂, 1 mMMgCl₂, 10 mM HEPES, 40 mM TEACl, 10 mM glucose, 87.5 mM CsCl (pH 7.2)and 108 mM CsMS, 4 mM MgCl₂, 9 mM EGTA, 9 mM HEPES (pH 7.2). Currentswere typically elicited from a holding potential of −80 mV to +10 mVusing Clampex software (Axon Instruments). Typically, currents werefirst elicited with low frequency stimulation (0.03 Hz) and allowed tostabilize prior to application of the compounds. The compounds were thenapplied during the low frequency pulse trains for two to three minutesto assess tonic block, and subsequently the pulse frequency wasincreased to 0.2 Hz to assess frequency dependent block. Data wereanalyzed using Clampfit (Axon Instruments) and SigmaPlot 4.0 (JandelScientific).

[0078] Specific data obtained for N-type channels are shown in Table 1below. As indicated by the data in Table 1, the most potent inhibitorsat higher frequencies were MC-34D, JM-G-10, 39-1-B4 and 39-45-3 shown inFIG. 1. However, all of the compounds tested appeared to be reasonablygood blockers at this frequency. TABLE 1 % Block Estimated IC₅₀ (100 nM)(μM) α_(1B) (N-type) 0.03 Hz 0.2 Hz 0.03 Hz 0.2 Hz MC-34D 47 73 0.120.05 JM-G-10 38 63 0.17 0.06 39-1-B4 34 72 0.2 0.04 SH-123A 19 55 0.470.09 SH-123B 9 45 1.27 0.13 SH-134 20 41 0.44 0.15 SH-136 14 44 0.630.14 39-45-3 50 79 0.1 0.03 39-36-1 Lomerizine 24 54 0.39 0.09 39-36-229 60 0.32 0.07

[0079] Tables 2 and 3 show the results of similar experiments conductedwith P/Q-type and L-type channels expressed in HEK 293 cells. Ingeneral, the IC₅₀ values for MC-34D, JM-G-10, 39-1-B4 and 39-45-3 werehigher than those exhibited with respect to N-type channels. TABLE 2 %Block Estimated IC₅₀ (100 nM) (μM) α_(1A) (P/Q-type) 0.03 Hz 0.2 Hz 0.03Hz 0.2 Hz MC-34D 31 56 0.35 0.08 JM-G-10 28 62 0.3 0.07 39-1-B4 31 520.65 0.12 SH-123A 29 61 0.26 0.07 SH-123B 16 44 0.55 0.13 SH-134 20 450.45 0.12 SH-136 18 42 0.52 0.15 39-45-3 27 58 0.41 0.09 39-36-1Lomerizine 25 57 0.46 0.07 39-36-2 35 52 0.31 0.11

[0080] TABLE 3 % Block Estimated IC₅₀ (100 nM) (μM) α_(1C) (L-type) 0.03Hz 0.2 Hz 0.03 Hz 0.2 Hz MC-34D 14 24 0.63 0.39 JM-G-10 7 15 1.17 0.339-1-B4 19 29 0.5 0.3 SH-123A 21 37 0.62 0.25 SH-123B 8 14 2.06 0.74SH-134 11 21 1.6 0.83 SH-136 4 8 2.8 1.4 39-45-3 15 26 0.74 0.37 39-36-1Lomerizine 26 44 0.29 0.15 39-36-2 11 22 2.1 0.4

[0081] These data are summarized in Table 4 which show the ratio of IC₅₀values for P:N and L:N channels. As shown, with respect to specificityfor L-type channels in particular, the four compounds mentioned aboveshow much higher affinity for N-type and P/Q-type versus L-typechannels. TABLE 4 0.2 Hz N P/Q L P:N L:N MC-34D 0.05 0.08 0.39 1.6:1 8:1JM-G-10 0.06 0.07 0.3 1.2:1 5:1 39-1-B4 0.04 0.12 0.3   3:1 8:1 SH-123A0.09 0.07 0.25 0.8:1 3:1 SH-123B 0.13 0.13 0.74   1:1 6:1 SH-134 0.150.12 0.83 0.8:1 6:1 SH-136 0.14 0.15 1.4 1.1:1 10:1  39-45-3 0.03 0.090.37   3:1 12:1  39-36-1 Lomerizine 0.09 0.07 0.15 0.8:1 1.7:1   39-36-20.07 0.11 0.4 1.6:1 5.7:1  

[0082] These results are shown graphically in FIGS. 2 and 3.

1. A method to treat pain in a subject which method comprisesadministering to a subject in need of such treatment a compound of theformula

or the salts thereof, wherein Cy represents cyclohexyl and Φ representsphenyl; Y is CH═CHΦ, CHΦ₂, Φ or Cy, X is trivalent straight-chainalkylene (3-10C) or trivalent straight-chain 1-alkenylene (3-10C)optionally substituted by oxo at the C adjacent N when n is 0 and Y isΦ₂CH; and is otherwise trivalent straight-chain alkylene (5-10C) ortrivalent straight-chain 1-alkenylene (5-10C) optionally substituted byoxo at the C adjacent N; Z is N, or CHNR¹ wherein R¹ is alkyl (1-6C);and n is 0-5; wherein each Φ and Cy independently may optionally besubstituted by alkyl (1-6C) or by halo, CF₃, OCF₃, NO₂, NR₂, OR, SR,COR, COOR, CONR₂, NROCR or OOCR where R is H or alkyl (1-4C), or twosubstituents may form a 5-7 membered ring with the proviso that thecompounds of formula (1) contain at least one aromatic moiety.
 2. Themethod of claim 1 wherein the compound of formula (1) is of the formula

wherein X, Y, Z and n are as defined, and each Φ may optionally besubstituted as set forth, in claim
 1. 3. The method of claim 2 wherein Yis CH═CHΦ.
 4. The method of claim 3 wherein X is CH(CH₂)_(m)CO orCH(CH₂)_(m+1) wherein m is 4-10.
 5. The method of claim 4 wherein m is4.
 6. The method of claim 3 wherein Z is N and n is 1-3.
 7. The methodof claim 6 wherein the compound of formula (1) is MC-34D or asubstituted form thereof.
 8. The method of claim 2 wherein Y is Cy. 9.The method of claim 8 wherein X is CH(CH₂)_(m)CO or CH(CH₂)_(m+1)wherein m is 4-10.
 10. The method of claim 9 wherein m is
 4. 11. Themethod of claim 2 wherein Z is CH₂NH and n is
 1. 12. The method of claim11 wherein the compound of formula (1) is JM-G-10 or a substituted formthereof.
 13. The method of claim 2 wherein Y is Φ₂CH.
 14. The method ofclaim 13 wherein X is CH(CH₂)_(l)CO or CH(CH₂)_(l+1), wherein l is 1-10.15. The method of claim 14 wherein l is
 1. 16. The method of claim 13wherein Z is N.
 17. The method of claim 16 wherein the compound offormula (1) is 39-1-B4 or a substituted form thereof.
 18. The method ofclaim 2 wherein n is 0 or 1 and Y is Φ.
 19. The method of claim 18wherein X is CH(CH₂)_(m+1) or CH(CH₂)_(m)CO wherein m is 4-10.
 20. Themethod of claim 19 wherein m is
 4. 21. The method of claim 20 whereinthe compound of formula (1) is compound 39-45-3 or a differentlysubstituted or unsubstituted form thereof.
 22. A pharmaceuticalcomposition for use in treating pain which composition comprises, inadmixture with a pharmaceutically acceptable excipient, a dosage amountof at least one compound of the formula

or salts thereof, wherein Cy represents cyclohexyl and Φ representsphenyl; Y is CH═CHΦ or Φ, X is trivalent straight-chain alkylene (3-10C)or trivalent straight-chain 1-alkenylene (3-10C) optionally substitutedby oxo at the C adjacent N when n is 0 and Y is Φ₂CH; and is otherwisetrivalent straight-chain alkylene (5-10C) or trivalent straight-chain1-alkenylene (5-10C) optionally substituted by oxo at the C adjacent N;Z is N or CHNR¹, wherein R¹ is alkyl (1-6C); and n is 0-5; wherein eachΦ and Cy independently may optionally be substituted by alkyl (1-6C) orby halo, CF₃, OCF₃, NO₂, NR₂, OR, SR, COR, COOR, CONR₂, NROCR or OOCRwhere R is H or alkyl (1-4C), or two substituents may form a 5-7membered ring with the proviso that the compounds of formula (1) containat least one aromatic moiety.
 23. The composition of claim 22 whereinthe compound of formula (1) is of the formula

wherein X, Y, Z and n are as defined in claim
 22. 24. The composition ofclaim 23 wherein Y is CH═CHΦ.
 25. The composition of claim 24 wherein Xis CH(CH₂)_(m)CO or CH(CH₂)_(m+1) wherein m is 4-10.
 26. The compositionof claim 25 wherein m is
 4. 27. The composition of claim 26 wherein Z isN and n is 1-3.
 28. The composition of claim 27 wherein the compound offormula (1) is MC-34D or a substituted form thereof.
 29. The compositionof claim 23 wherein Z is CH₂NH and n is
 1. 30. The composition of claim29 wherein the compound of formula (1) is JM-G-10 or a substituted formthereof.
 31. The composition of claim 23 wherein n is 0 or 1 and Y is Φ.32. The composition of claim 31 wherein X is CH(CH₂)_(m+1) orCH(CH₂)_(m)CO wherein m is 4-10.
 33. The composition of claim 32 whereinm is
 4. 34. The composition of claim 33 wherein the compound of formula(1) is compound 39-45-3 or a differently substituted or unsubstitutedform thereof.